*Target (protein/gene name): Phospholipase C (alpha toxin) *NCBI Gene # or RefSeq#:4201274 *Protein ID (NP or XP #) or Wolbachia#:2WY6 *Organism (including strain):clostridium perfrigens Etiologic Risk Group (see link below): */Disease Information(sort of like the Intro to your MiniResearch Writeup): Clostridium perfringens alpha toxin is atoxin produced by thebacteriumClostridium perfringens (C. perfringens) and is responsible forgas gangrene andmyonecrosis ininfectedtissues. Link to TDR Targets page (if present):None Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Complex of proteins?:monomer Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): *EC#:3.1.4.3 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.4.3&Suchword=&organism%5B%5D=Clostridium+perfringens -- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assayinformation (spectrophotometric, coupled assay ?, reagents):colorimetric -- link to Sigma page for assay http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/phospholipaseccper.pdf --- List cost and quantity of substrate reagents, supplier, and catalog # Phospholipase C from clostridium perfringens P7633-25UN; CAS: 0009001869; $75.10 (Sigma) Alkaline Phosphatase 10713023001; CAS:0000000000; $91.99 (Sigma) Structure (PDB or Homology model) -- PDB #: 2WY6 Current Inhibitors:21 Expression Information (has it been expressed in bacterial cells): Alpha toxin and N-terminal domain expressed in Escherichia coli Purification Method: Affinity chromatography, gel filtration, 2500fold purification from culture filtrate Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence WDGKIDGTGTHAMIVTQGVSILENDLSKNEPESVRKNLEILKENMHELQLGSTYPDYDKNAYDLYQDHFWDPDIDNNFSK DNSWYLAYSIPDTGESQIRKFSALARYEWQRGNYKQATFYLGEAMHYFGDIDTPYHPANVTAVDSAGHVKFETFAEERKE QYKINTAGCKTNEAFYTDILKNKDFNAWSKEYARGFAKTGKSIYYSHASMSHSWDDWDYAAKVTLANSQKGTAGYIYRFL HDVSEGNDPSVGKNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMTGSKDTYTFKLKDENLKIDD IQNMWIRKRKYTAFPDAYKPENIKIIANGKVVVDKDINEWISGNSTYNIK
*length of your protein in Amino Acids:370 Molecular Weightof your protein in kiloDaltons using the**Expasy ProtParam**website42546.0 MolarExtinction coefficientof your protein at 280 nm wavelength:93740 M-1 cm-1 at 280nm TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDSGene Sequence(paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): *GC% Content for gene (codon optimized): Do Not Need this info forSpring(but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works outputtext file-that should be saved in yourGoogle Docsfolder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file ifyou are notsure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#:4201274
*Protein ID (NP or XP #) or Wolbachia#:2WY6
*Organism (including strain): clostridium perfrigens
Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Clostridium perfringens alpha toxin is a toxin produced by thebacterium Clostridium perfringens (C. perfringens) and is responsible for gas gangrene and myonecrosis in infected tissues.
Link to TDR Targets page (if present): None
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein:
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Complex of proteins?: monomer
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 3.1.4.3
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.4.3&Suchword=&organism%5B%5D=Clostridium+perfringens
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): colorimetric
-- link to Sigma page for assay
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/phospholipaseccper.pdf
--- List cost and quantity of substrate reagents, supplier, and catalog #
Phospholipase C from clostridium perfringens
P7633-25UN; CAS: 0009001869; $75.10 (Sigma)
Alkaline Phosphatase
10713023001; CAS:0000000000; $91.99 (Sigma)
Structure (PDB or Homology model)
-- PDB #: 2WY6
Current Inhibitors: 21
Expression Information (has it been expressed in bacterial cells):
Alpha toxin and N-terminal domain expressed in Escherichia coli
Purification Method:
Affinity chromatography, gel filtration, 2500fold purification from culture filtrate
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence
WDGKIDGTGTHAMIVTQGVSILENDLSKNEPESVRKNLEILKENMHELQLGSTYPDYDKNAYDLYQDHFWDPDIDNNFSK
DNSWYLAYSIPDTGESQIRKFSALARYEWQRGNYKQATFYLGEAMHYFGDIDTPYHPANVTAVDSAGHVKFETFAEERKE
QYKINTAGCKTNEAFYTDILKNKDFNAWSKEYARGFAKTGKSIYYSHASMSHSWDDWDYAAKVTLANSQKGTAGYIYRFL
HDVSEGNDPSVGKNVKELVAYISTSGEKDAGTDDYMYFGIKTKDGKTQEWEMDNPGNDFMTGSKDTYTFKLKDENLKIDD
IQNMWIRKRKYTAFPDAYKPENIKIIANGKVVVDKDINEWISGNSTYNIK
*length of your protein in Amino Acids: 370
Molecular Weight of your protein in kiloDaltons using the **Expasy ProtParam** website42546.0
Molar Extinction coefficient of your protein at 280 nm wavelength: 93740 M-1 cm-1 at 280nm
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**